Project 1, entitled Immunogen Design and Selection, plays the major role of generating the HIV envelope glycoprotein-based candidate vaccine immunogens to be analyzed in this HIVRAD. In Aim 1, we will design novel trimeric immunogens based upon three distinct strategies. Initially, we will utilize recent mapping data performed in our collaborative group on CD4 binding-site (CD4bs) directed neutralizing antibodies (nAbs) isolated from non-human primates (NHPs) immunized with HIV Env trimeric immunogens. We compared the NHP CD4bs nAbs to the known, more broadly nAbs isolated from HIV-1-infected human subjects t)y alanine scanning mutagenesis ofthe HlV-1 gpl 20. Using the alanine scan information, we generated divergent mAb footprint patterns between the NHP CD4bs Mabs and the structurally defined footprint ofthe human bNabs. Armed with this information, we will design stabilized trimers that preferentially present neutralizing epitopes to activate VH genes associated with the infection-elicited bNabs and to elicit neutralizing antibody responses by vaccination. In the 2nd approach, we will assess the ability of loop-deleted, conformationally stabilized trimers, which more rapidly elicit CD4bs-directed in rabbits, to do so in NHPs. Improved versions will be assessed for their capacity to elicit CD4bs Mabs or to synergize with full-length trimers to elicit CD4bs-directed B cell responses. In Aim 2, we will generate sFVs of our isolated NHP CD4bs Abs (GE148 or GE136) and perform yeast cell-surface display, random mutagenesis and selection with conformationally stabilized Env core antigens to determine if we can evolve these non-broadly neutralizing CD4bs mAbs to become more broadly neutralizing and the levels of mutation required to achieve such a capacity. Finally, in Aim 3, we will select novel Env trimeric immunogens using directed evolution as a complementary approach to rational Env design. Env trimers, stabilized in the context of infectious virus using selection criteria established by Dr. Michael Zwick, will be assessed for their ability to elicit B cell responses in NHPs. In addition, HIV-1 Env trimers will be selected in the Zwick laboratory for recognition by NHP germline equivalents of CD4bs bNabs (e.g. VH1-02) or PG9 or PGT128 by iterative affinity-selection of viral mutants.